Identification of Critical Amino Acid Residues of a Two-Component Sensor Protein for Signal Sensing in Porphyromonas gingivalis Fimbriation via Random Mutant Library Construction.

Porphyromonas gingivalis (Pg) utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS-FimR two-component system, making the FimS sensory protein an attractive target for preventing Pg infection. However, the specific environmental signal received by FimS remains unknown. We constructed random Pg mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of fimS, and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the fimS-knockout Pg strain using the Escherichia coli-Pg conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased fimA expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of Pg, including the elucidation of structure-function relationships of proteins of interest.

Thermostability Enhancement of GH 62 α-l-Arabinofuranosidase by Directed Evolution and Rational Design.

GH 62 arabinofuranosidases are known for their excellent specificity for arabinoxylan of agroindustrial residues and their synergism with endoxylanases and other hemicellulases. However, the low thermostability of some GH enzymes hampers potential industrial applications. Protein engineering research highly desires mutations that can enhance thermostability. Therefore, we employed directed evolution using one round of error-prone PCR and site-saturation mutagenesis for thermostability enhancement of GH 62 arabinofuranosidase from Aspergillus fumigatus. Single mutants with enhanced thermostability showed significant ΔΔG changes (<-2.5 kcal/mol) and improvements in perplexity scores from evolutionary scale modeling inverse folding. The best mutant, G205K, increased the melting temperature by 5 °C and the energy of denaturation by 41.3%. We discussed the functional mechanisms for improved stability. Analyzing the adjustments in α-helices, ß-sheets, and loops resulting from point mutations, we have obtained significant knowledge regarding the potential impacts on protein stability, folding, and overall structural integrity.

Production of octyl butyrate using psychrophilic mutant lipase from Croceibacter atlanticus LipCA lipase developed by a molecular evolution technique.

Lipases are used to synthesize a variety of industrially useful compounds. Among them, psychrophilic lipase can be used to synthesize thermo-labile compounds at low temperatures. In this study, random mutagenesis was introduced into Antarctic Croceibacter atlanticus lipase gene using error-prone PCR, resulting in changes in its protein sequence. Through two rounds of mutagenesis and screening, we found that a mutant R1 showed an enhanced activity at low temperatures. Mutant R1 had five mutations (F43L, S48G, S49G, D141K, and K297R) and higher kcat/KM value than the wild type (WT) at 10 °C. We immobilized this enzyme on methacrylate divinylbenzene resin and used it to synthesize octyl butyrate, a flavor compound. The esterification reaction proceeded even at 10 °C. Mutant R1 synthesized the ester compound faster than the WT. To determine which amino acids were responsible for the increase of activity, site-directed mutagenesis was performed to introduce five back mutations into mutant R1. Three back mutants (L43F, G48S, G49S) showed significant decreases of activity at low temperatures, indicating that these amino acids were closely related to the increase in activity. This psychrophilic mutant R1 is expected to be used in low-temperature enzyme conversion reactions in the food industry.

Rational and random mutagenesis of GDEst-95 carboxylesterase: New functionality insights.

Lipolytic enzymes are important contributors in industrial processes from lipid hydrolysis to biofuel production or even polyester biodegradation. While these enzymes can be used in numerous applications, the genotype-phenotype space of certain promising enzymes is still poorly explored. This limits the effective application of such biocatalysts. In this work the genotype space of a 55 kDa carboxylesterase GDEst-95 from Geobacillus sp. 95 was explored using site-directed mutagenesis and directed evolution methods. In this study four site-directed mutants (Gly108Arg, Ala410Arg, Leu226Arg, Leu411Ala) were created based on previous analysis of GDEst-95 carboxylesterase. Error-prone PCR resulted three mutants: two of them with distal mutations: GDEst-RM1 (Arg75Gln), GDEst-RM2 (Gly20Ser Arg75Gln) and the third, GDEst-RM3, with a distal (Ser210Gly) and Tyr317Ala (amino acid position near to the active site) mutation. Mutants with Ala substitution displayed approximately twofold higher specific activity. Arg mutations lead a reduced specific activity, retaining 2.86 % (Gly108Arg), 10.95 % (Ala410Arg), and 44.23 % (Leu226Arg) of lipolytic activity. All three random mutants displayed increased specific activity as well as improved catalytic properties. This research provides the first deeper insights into the functionality of understudied Geobacillus spp. carboxylesterases with 55 kDa in size.

Improved thermostability of type I pullulanase from Bacillus thermoliquefaciens by error-prone PCR.

Pullulanase (PulB) is a starch-debranching enzyme. In order to improve its catalytic performance, random mutagenesis was performed on the pullulanase gene derived from Bacillus thermoliquefaciens. Two rounds of error-prone PCR were carried out. Mutant T252S was screened in the first round of error-prone library, which had the highest catalytic activity. During the second round of mutations, mutant enzyme G250P/T252S/G253T/N255K was screened, which had further improved catalytic activity and the best thermostability. Compared with the parent enzyme, the specific activity of mutant enzyme G250P/T252S/G253T/N255K increased by 1.9 times, Km decreased by 22.7 %, kcat increased by 28.7 %, and kcat/Km increased by 68.4 %. The thermostability of the mutant enzyme improved significantly, showing that the half-life at 60 °C was extended to 7.5 h, which was 87.5 % higher than that of the parent enzyme. The mutation sites in these two rounds were concentrated in the 250-255 regions, indicating that this region was an important region affecting the catalytic activity and Thermostability. The reasons for the change of enzymtic properties was also preliminarily analyzed through three-dimensional simulation.

Directed evolution of cholesterol oxidase with improved thermostability using error-prone PCR.

Cholesterol oxidase is industrially important as it is frequently used as a biosensor in food and agriculture industries and measurement of cholesterol. Although, most natural enzymes show low thermostability, which limits their application. Here, we obtained an improved variant of Chromobacterium sp. DS1 cholesterol oxidase (ChOS) with enhanced thermostability by random mutant library applying two forms of error-prone PCR (serial dilution and single step). Wild-type ChOS indicated an optimal temperature and pH of 70 ºC and pH 7.5, respectively. The best mutant ChOS-M acquired three amino acid substitutions (S112T, I240V and A500S) and enhanced thermostability (at 50 °C for 5 h) by 30%. The optimum temperature and pH in the mutant were not changed. In comparison to wild type, circular dichroism disclosed no significant secondary structural alterations in mutants. These findings show that error-prone PCR is an effective method for enhancing enzyme characteristics and offers a platform for the practical use of ChOS as a thermal-resistance enzyme in industrial fields and clinical diagnosis.

Tuning Thermostability and Catalytic Efficiency of Aflatoxin-Degrading Enzyme by Error-prone PCR.

In our previous work, a recombinant aflatoxin-degrading enzyme derived from Myxococcus fulvus (MADE) was reported. However, the low thermal stability of the enzyme had limitations for its use in industrial applications. In this study, we obtained an improved variant of recombinant MADE (rMADE) with enhanced thermostability and catalytic activity using error-prone PCR. Firstly, we constructed a mutant library containing over 5000 individual mutants. Three mutants with T50 values higher than the wild-type rMADE by 16.5 °C (rMADE-1124), 6.5 °C (rMADE-1795), and 9.8 °C (rMADE-2848) were screened by a high-throughput screening method. Additionally, the catalytic activity of rMADE-1795 and rMADE-2848 was improved by 81.5% and 67.7%, respectively, compared to the wild-type. Moreover, structural analysis revealed that replacement of acidic amino acids with basic amino acids by a mutation (D114H) in rMADE-2848 increased the polar interactions with surrounding residues and resulted in a threefold increase in the t1/2 value of the enzyme and made it more thermaltolerate. KEY POINTS: • Mutant libraries construction of a new aflatoxins degrading enzyme by error-prone PCR. • D114H/N295D mutant improved enzyme activity and thermostability. • The first reported enhanced thermostability of aflatoxins degrading enzyme better for its application.

Directed Evolution of the BpsA Carrier Protein Domain for Recognition by Non-cognate 4'-Phosphopantetheinyl Transferases to Enable Inhibitor Screening.

4'-Phosphopantetheinyl transferases (PPTases) play an essential role in activating the carrier protein domains of mega-synthases involved in primary and secondary metabolism and have been validated as promising drug targets in multiple pathogens. Monitoring phosphopantetheinylation of the non-ribosomal peptidase synthetase BpsA, which produces blue indigoidine pigment upon activation, is a useful strategy to screen chemical collections for inhibitors of a target PPTase. However, PPTases can exhibit carrier protein specificity and some medically important PPTases do not activate BpsA. Here, we describe how to conduct a directed evolution campaign to evolve the BpsA carrier protein domain for improved recognition by a candidate PPTase, as exemplified for the human Sfp-like PPTase. This method can be applied to other non-cognate PPTases for discovery of new drug candidates or chemical probes, or to enable development of next-generation biosensors that utilize BpsA as a reporter.

Thermostability enhancement of Escherichia coli phytase by error-prone polymerase chain reaction (epPCR) and site-directed mutagenesis.

Phytase efficiently hydrolyzes phytate to phosphate; thus, it is widely used to increase phosphorus availability in animal feeds and reduce phosphorus pollution through excretion. Phytase is easily inactivated during feed pelleting at high temperature, and sufficient thermostability of phytase is essential for industrial applications. In this study, directed evolution was performed to enhance phytase thermostability. Variants were initially expressed in Escherichia coli BL21 for screening, then in Pichia pastoris for characterization. Over 19,000 clones were generated from an error-prone Polymerase Chain Reaction (epPCR) library; 5 mutants (G10, D7, E3, F8, and F9) were obtained with approximately 9.6%, 10.6%, 11.5%, 11.6%, and 12.2% higher residual activities than the parent after treatment at 99°C for 60 min. Three of these mutants, D7, E3, and F8, exhibited 79.8%, 73.2%, and 92.6% increases in catalytic efficiency (kcat/Km), respectively. In addition, the specific activities of D7, E3, and F8 were 2.33-, 1.98-, and 2.02-fold higher than parental phytase; they were also higher than the activities of all known thermostable phytases. Sequence analysis revealed that all mutants were substituted at residue 75 and was confirmed that the substitution of cysteine at position 75 was the main contribution to the improvement of thermostability of mutants by saturation mutagenesis, indicating that this amino acid is crucial for the stability and catalytic efficiency of phytase. Docking structure analysis revealed that substitution of the C75 residue allowed the mutants to form additional hydrogen bonds in the active pocket, thereby facilitating binding to the substrate. In addition, we confirmed that the intrinsic C77-C108 disulfide bond in E. coli phytase is detrimental to its stability.

Enhancing the Hydrolysis and Acyl Transfer Activity of Carboxylesterase DLFae4 by a Combinational Mutagenesis and In-Silico Method.

In the present study, a feruloyl esterase DLFae4 identified in our previous research was modified by error-prone PCR and site-directed saturation mutation to enhance the catalytic efficiency and acyltransferase activity further. Five mutants with 6.9-118.9% enhanced catalytic activity toward methyl ferulate (MFA) were characterized under the optimum conditions. Double variant DLFae4-m5 exhibited the highest hydrolytic activity (270.97 U/mg), the Km value decreased by 83.91%, and the Kcat/Km value increased by 6.08-fold toward MFA. Molecular docking indicated that a complex hydrogen bond network in DLFae4-m5 was formed, with four of five bond lengths being shortened compared with DLFae4, which might account for the increase in catalytic activity. Acyl transfer activity assay revealed that the activity of DLFae4 was as high as 1550.796 U/mg and enhanced by 375.49% (5823.172 U/mg) toward 4-nitrophenyl acetate when residue Ala-341 was mutated to glycine (A341G), and the corresponding acyl transfer efficiency was increased by 7.7 times, representing the highest acyltransferase activity to date, and demonstrating that the WGG motif was pivotal for the acyltransferase activity in family VIII carboxylesterases. Further experiments indicated that DLFae4 and variant DLFae4 (A341G) could acylate cyanidin-3-O-glucoside effectively in aqueous solution. Taken together, our study suggested the effectiveness of error-prone PCR and site-directed saturation mutation to increase the specific activity of enzymes and may facilitate the practical application of this critical feruloyl esterase.

Structural and functional analyses of GH51 alpha-L-arabinofuranosidase of Geobacillus vulcani GS90 reveal crucial residues for catalytic activity and thermostability.

Alpha-L-arabinofuranosidase (Abf) is of big interest in various industrial areas. Directed evolution is a powerful strategy to identify significant residues underlying Abf properties. Here, six active variants from GH51 Abf of Geobacillus vulcani GS90 (GvAbf) by directed evolution were overproduced, extracted, and analyzed at biochemical and structural levels. According to the activity and thermostability results, the most-active and the least-active variants were found as GvAbf51 and GvAbf52, respectively. GvAbf63 variant was more active than parent GvAbf by 20% and less active than GvAbf51. Also, the highest thermostability belonged to GvAbf52 with 80% residual activity after 1 h. Comparative sequence and structure analyses revealed that GvAbf51 possessed L307S displacement. Thus, this study suggested that L307 residue may be critical for GvAbf activity. GvAbf63 had H30D, Q90H, and L307S displacements, and H30 was covalently bound to E29 catalytic residue. Thus, H30D may decrease the positive effect of L307S on GvAbf63 activity, preventing E29 action. Besides, GvAbf52 possessed S215N, L307S, H473P, and G476C substitutions and S215 was close to E175 (acid-base residue). S215N may partially disrupt E175 action. Overall effect of all substitutions in GvAbf52 may result in the formation of the C-C bond between C171 and C213 by becoming closer to each other.

Directed Evolution of Fluorescent Proteins in Bacteria.

Directed evolution has revolutionized the way scientists create new biomolecules not found in nature. Error-prone polymerase chain reaction (PCR) introduces random mutations and was used to evolve jellyfish and coral fluorescent proteins in bacteria. We describe a novel method for the directed evolution of a far-red fluorescent protein in E. coli. The new method used genes to produce fluorophores inside E. coli and allowed changing the native fluorophore, phycocyanobilin, for a second small-molecule fluorophore, biliverdin. The directed evolution blueshifted the fluorescence, which enhanced the quantum yield to produce a brighter fluorescent protein. Finally, the evolution selected fluorescent proteins that expressed in large quantities in E. coli. The evolved fluorescent protein was named the small ultra-red fluorescent protein (smURFP) and was biophysically as bright as the enhanced green fluorescent protein (EGFP). This chapter describes the materials and methods used to evolve a far-red fluorescent protein in bacteria. While the focus is a fluorescent protein, the protocol is adaptable for the evolution of other biomolecules in bacteria when using a proper selection strategy.

Yeast Surface Display Platform for Rapid Selection of an Antibody Library via Sequential Counter Antigen Flow Cytometry.

Yeast surface display techniques have been increasingly employed as a tool for both the discovery and affinity maturation of antibodies. In this study, we describe the use of yeast surface display for the selection and affinity maturation of antibodies targeted to small molecules (haptens). In this approach, we coupled 4 to 15 sequential cycles of error-prone PCR to introduce heterogeneity into the sequence of an 12F6 scFv antibody that binds to chelated uranium; the resulting full-length constructs were combined to create a yeast-displayed scFv-library with high diversity. We also developed a stringent selection technique utilizing fluorescence-activated cell sorting; this was based on sequentially dropping the target antigen concentration, while concomitantly increasing the concentration of potential cross-reactive haptens in subsequent selection cycles. As a proof of the efficacy this approach, we confirmed that the antibodies identified via this approach retained binding to the target antigen (UO22+ complexed to a chelator), while binding with lesser affinity than the parental scFv to a structurally related haptens (the same chelator complexed to other metal ions). As will be described in this report, these scFv variants perform more efficiently in sensor-based assay than the parental 12F6 antibody. Combining the generation of scFv libraries via error-prone PCR with selection of yeast-displayed antibodies by fluorescence activated cell sorting will provide an efficient new method for the isolation of scFvs and other binding proteins with high affinity and specificity.

Evolutionary approach for improved proton pumping activity of heterologous rhodopsin expressed in Escherichia coli.

A light-driven ATP regeneration system using rhodopsin has been utilized as a method to improve the production of useful substances by microorganisms. To enable the industrial use of this system, the proton pumping rate of rhodopsin needs to be enhanced. Nonetheless, a method for this enhancement has not been established. In this study, we attempted to develop an evolutionary engineering method to improve the proton-pumping activity of rhodopsins. We first introduced random mutations into delta-rhodopsin (dR) from Haloterrigena turkmenica using error-prone PCR to generate approximately 7000 Escherichia coli strains carrying the mutant dR genes. Rhodopsin-expressing E. coli with enhanced proton pumping activity have significantly increased survival rates in prolonged saline water. Considering this, we enriched the mutant E. coli cells with higher proton pumping rates by selecting populations able to survive starvation under 50 µmol m-2 s-1 at 37 °C. As a result, we successfully identified two strains, in which proton pumping activity was enhanced two-fold by heterologous expression in E. coli in comparison to wild-type strains. The combined approach of survival testing using saline water and evolutionary engineering methods used in this study will contribute greatly to the discovery of a novel rhodopsin with improved proton pumping activity. This will facilitate the utilization of rhodopsin in industrial applications.

Isolation and Characterization of Engineered Nucleoside Deoxyribosyltransferase with Enhanced Activity Toward 2'-Fluoro-2'-Deoxynucleoside.

Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDTL59Q, showed 1.7-fold (at 40°C) and 4.4-fold (at 50°C) higher 2FDU-to-2FDA conversions compared to the NDTWT, respectively. Subsequently, both NDTWT and NDTL59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDTL59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

Determination of Nucleotide Sequences within Promoter Regions Affecting Promoter Compatibility between Zymomonas mobilis and Escherichia coli.

A promoter plays a crucial role in controlling the expression of the target gene in cells, thus being one of the key biological parts for synthetic biology practices. Although significant efforts have been made to identify and characterize promoters with different strengths in various microorganisms, the compatibility of promoters within different hosts still lacks investigation. In this study, we chose the native Pgap promoter of Zymomonas mobilis to investigate nucleotide sequences within promoter regions affecting promoter compatibility between Escherichia coli and Z. mobilis. Pgap is one of the strongest promotors in Z. mobilis that has many excellent characteristics to be developed as microbial cell factories. Using EGFP as a reporter, a Z. mobilis-derived Pgap mutant library was constructed and sorted in E. coli, with candidate promoters exhibiting high fluorescence intensity collected. A total of 53 variants were finally selected and sequenced by Sanger sequencing. The sequencing results grouped these variants into 12 different Pgap variant types, among which seven types presented higher promoter strength than native Pgap in E. coli. The next-generation sequencing technique was then employed to identify key mutations within the Pgap promoter region that affect the promoter compatibility. Finally, six important sites were identified and confirmed to help increase Pgap strength in E. coli while keeping similar strength of native Pgap in Z. mobilis. Compared to native Pgap, synthetic promoters combining these sites had enhanced strength; especially, Pgap-6M combining all six sites exhibited 20-fold greater strength than native Pgap in E. coli. This study thus not only determined six important sites affecting promoter compatibility but also confirmed a series of Pgap promoter variants with strong promoter activity in both E. coli and Z. mobilis. In addition, a strategy was established in this study to investigate and determine nucleotide sequences in promoter regions affecting promoter compatibility, which can be applied in other microorganisms to help reveal universal factors affecting promoter compatibility and design promoters with desired strengths among different microbial cell factories.

Directed evolution of maltogenic amylase from Bacillus licheniformis R-53: Enhancing activity and thermostability improves bread quality and extends shelf life.

Maltogenic amylase from Bacillus licheniformis R-53 improves bread quality and shelf life. Here, we constructed a random mutation library of this enzyme using error-prone PCR to identify mutants with higher activity and thermostability, then screened the key point mutations. Mutant V296F/K418I had 2.16 times the specific activity of the wild-type enzyme, and its temperature for optimum activity increased from 60 to 65 °C. Moreover, it maintained > 60% residual activity after heating at 55 to 80 °C for 30 min. Mixolab experiments showed that treatment with 45 ppm V296F/K418I mutant maltogenic amylase had better effects in delaying dough recrystallization than 60 ppm wild-type enzyme. During bread storage, 45 ppm V296F/K418I was more effective in reducing hardness, improving elasticity, and maintaining sensory than 60 ppm wild-type enzyme. Directed evolution of maltogenic amylase significantly improved its activity and thermostability; the mutant enzyme is conducive to improving bread quality and extending its shelf life.

Improvement of thermostability of cholesterol oxidase from streptomyces Sp. SA-COO by random mutagenesis.

To enhance the thermal stability of Streptomyces Sp. SA-COO cholesterol oxidase, random mutagenesis was used. A random mutant library was generated using two types of error-prone PCR (single step and serial dilution) and two mutants (ChOA-M1 and ChOA-M2) with improved thermostability were obtained. The best mutant ChOA-M1 acquired three amino acid substitutions (G49T, W52K, and F62V) and improved thermostability (at 50 °C for 5 h) by 40% and increased the kcat/Km value by 23%. The optimum pH was desirably changed to encompass a broad range from alkali to acid and circular dichroism revealed no significant secondary structure changes in mutants against wild type. These findings indicated that random mutagenesis was an effective technique for optimizing cholesterol oxidase properties and make a foundation for practical applications of Cholesterol oxidase in clinical diagnosis and industrial fields.

Engineering Ag43 Signal Peptides with Bacterial Display and Selection.

Protein display, secretion, and export in prokaryotes are essential for utilizing microbial systems as engineered living materials, medicines, biocatalysts, and protein factories. To select for improved signal peptides for Escherichia coli protein display, we utilized error-prone polymerase chain reaction (epPCR) coupled with single-cell sorting and microplate titer to generate, select, and detect improved Ag43 signal peptides. Through just three rounds of mutagenesis and selection using green fluorescence from the 56 kDa sfGFP-beta-lactamase, we isolated clones that modestly increased surface display from 1.4- to 3-fold as detected by the microplate plate-reader and native SDS-PAGE assays. To establish that the functional protein was displayed extracellularly, we trypsinized the bacterial cells to release the surface displayed proteins for analysis. This workflow demonstrated a fast and high-throughput method leveraging epPCR and single-cell sorting to augment bacterial surface display rapidly that could be applied to other bacterial proteins.

Directed evolution driving the generation of an efficient keratinase variant to facilitate the feather degradation.

Keratinases can specifically degrade keratins, which widely exist in hair, horns, claws and human skin. There is a great interest in developing keratinase to manage keratin waste generated by the poultry industry and reusing keratin products in agriculture, medical treatment and feed industries. Degradation of keratin waste by keratinase is more environmentally friendly and more sustainable compared with chemical and physical methods. However, the wild-type keratinase-producing strains usually cannot meet the requirements of industrial production, and some are pathogenic, limiting their development and utilization. The main purpose of this study is to improve the catalytic performance of keratinase via directed evolution technology for the degradation of feathers. We first constructed a mutant library through error-prone PCR and screened variants with enhanced enzyme activity. The keratinase activity was further improved through fermentation conditions optimization and fed-batch strategies in a 7-L bioreactor. As a result, nine mutants with enhanced activity were identified and the highest enzyme activity was improved from 1150 to 8448 U/mL finally. The mutant achieved efficient biodegradation of feathers, increasing the degradation rate from 49 to 88%. Moreover, a large number of amino acids and soluble peptides were obtained as degradation products, which were excellent protein resources to feed. Therefore, the study provided a keratinase mutant with application potential in the management of feather waste and preparation of protein feed additive.